Sexual reproduction provides ample opportunity for pairwise interactions between the genotypes of mating pairs. One consequence of processes that are rife with male x female interactions is that evolution may fail to optimize functions for either sex, and instead there is retained a high level of variation. This appears to be the case for factors influencing sperm competition and other reproductive fitness attributes in Drosophila. The seminal proteins known as accessory gland proteins (Acps) appear to be specialized for mediating aspects of these interactions, and along with the female molecules that interact with Acps, they serve as a molecular entr[unreadable] to quantifying specific male x female interactions. Our first specific aim is to use genetic isolates from natural populations to quantify male x female interactions in components of reproductive fitness with a focus on those likely to be mediated by Accessory Gland Proteins and the proteins processes with which they interact in females. Using lines from the Drosophila Genetic Reference Panel (for which both genome sequence and gene expression data are already available), supplemented where appropriate with additional lines from five broad geographic regions, we will examine male x female interactions both within and between lines and regions in a 20 x 20 partial diallel design. Paternity success in doubly-mated females, fecundity, fertility, and female remating receptivity will all be systematically scored. This study will quantify reproductive fitness components for validation of the findings of Aims 2 and 3. The second aim is to dissect male x female interactions by testing RNAi knockdowns of specific male Acp genes across a range of well-characterized wild female genotypes. Comparison of the same reproductive phenotypes will be scored using males that express Acps normally, or are knocked down by RNAi for specific Acps. We will begin with a focus on Acps that have been associated with effects on sperm storage and/or sperm competition. In addition, we will examine a specific test case in which both the male and female partners are known, by testing for interactions among variant alleles in Sex Peptide and the Sex Peptide receptor. Female lines that are hyper- and hyporesponsive to each knockdown will be identified, and we will partition total interaction into components mediated by each Acp. By extending our understanding of the genetic variation in male and female contributors to reproduction, we will obtain insights into how and why genetic variation is maintained in nature. Moreover, since the genes whose variation we examine involve basic reproductive functions, our results will expand the foundation for understanding cases of idiopathic human infertility, specifically those in which no sex-specific cause for the infertility can be identified and may thus reflect genetic incompatibility between the partners.